Characterization of two whey protein genes in the Australian dasyurid marsupial, the stripe-faced dunnart <i>(Sminthopsis macroura)</i>

Abstract

We report the first isolation and sequencing of genomic BAC clones containing the marsupial milk protein genes Whey Acidic Protein (WAP) and Early Lactation Protein (ELP). The stripe-faced dunnart WAPgene sequence contained five exons, the middle three of which code for the WAPmotifs and four disulphide core domains which characterize WAP. The dunnart ELPgene sequence contained three exons encoding a protein with a Kunitz motif common to serine protease inhibitors. Fluorescence in situ hybridization located the WAPgene to chromosome 1p in the stripe-faced dunnart, and the ELPgene to 2q. Northern blot analysis of lactating mammary tissue of the closely related fat-tailed dunnart has shown asynchronous expression of these milk protein genes. ELPwas expressed at only the earlier phase of lactation and WAPonly at the later phase of lactation, in contrast to β-lactoglobulin (BLG) and α-lactalbumin (ALA) genes, which were expressed in both phases of lactation. This asynchronous expression during the lactation cycle in the fat-tailed dunnart is similar to other marsupials and it probably represents a pattern that is ancestral to Australian marsupials.

Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene; new insights into the function of the protein

“…The structure of the tWAP gene consists of five exons and is similar to the gene from stripe‐faced dunnart, brush‐tailed possum, and gray short‐tailed opossum (Simpson et al 2000; De Leo et al 2006). The WAP from each of the marsupials encodes a leader peptide sequence in exon 1, exon 2 codes for DIII (marsupial specific), exon 3 for DI, whereas DII is encoded by exon 4 (Simpson et al 2000).…”

“…In contrast, eutherian WAPs are characterized by four exons, where exon 2 codes for DI, and exon 3 for DII. The WAP exon–intron size and sequence is well conserved between marsupial species such as tammar wallaby, brush‐tailed possum, and stripe‐faced dunnart, with the size of their primary transcript region ranging between 5.2 and 6.2 kb (De Leo et al 2006). On the other hand, eutherian WAP genes are significantly smaller ranging between 1.7 and 3.1 kb in length, and exhibiting greater exon–intron size variation between species (Campbell et al 1984; Thepot et al 1990; Rival‐Gervier et al 2003).…”

“…Marsupial WAPs are characterized by three domains, with the WAP motif (KXGCP) present in marsupial domains I, II, and III (DI, DII, and DIII, respectively). The WAP gene from three marsupials has been sequenced and is composed of five exons (Simpson et al 2000; De Leo et al 2006), with exon 2 encoding a marsupial‐specific DIII. The WAP gene has also been described in some other species including humans (Clauss et al 2002) and ruminants (ewe, goat, and cow; Hajjoubi et al 2006).…”

The mammary gland-specific marsupial ELP and eutherian CTI share a common ancestral gene

“…We used fluorescence in situ hybridisation to map tammar ELP to chromosome 1q (Figure 3). The ELP/CTI gene was located on a syntenic segment in the marsupial (stripe-faced dunnart [27] and opossum) and eutherian genomes [49,55] and was generally flanked by one or both of the single-copy genes phosphatidyl inositol glycan, class T ( PIGT ) and WAP four disulphide core domain 2 ( WFDC2 ), confirming they were true orthologues (Figure 4). …”

“…ELP was later identified in the tammar [13,20,21,26], the stripe-faced and fat-tailed dunnarts ( Sminthopsis macroura and Sminthopsis crassicaudata respectively) and the South American grey short-tailed opossum ( Monodelphis domestica ) [27] (Refer to Additional file 1: Table S1 for the species in which the putative functional ELP / CTI gene, transcript and protein have been identified). Marsupial ELP expression is limited to the early phase of lactation [13,20,21,27,28] at the time the mother produces milk for an immunologically naïve young [29,30]. During this period, the tammar young is permanently attached to the teat and protected by humoral (passive) immunity acquired from its mother’s milk and its own innate immunity [18,30].…”